Project Summary Enabling high-accuracy nanopore-based strand sequencing has the potential to improve the understanding of long-range genomic relationships and processes and, based on its accessibility, enable the associated clinical and diagnostic applications (the fastest growing sequencing sector). Unfortunately, it is commonplace for nanopore sequencing error rates to exceed 4%, and higher accuracy is needed to advance nanopore sequencing applications toward assay-specific applications. Among the various DNA sequencing technologies, nanopore strand sequencing has emerged as a very promising method to enable genomics, transcriptomics, epigenomics, and epitranscriptomics due to the currently associated advantages, such as adaptability, cost, direct-read capabilities, read length, etc., relative to presently market-dominating sequencing-by-synthesis technologies. However, the library preparation procedures associated with nanopore-based strand sequencing approaches, along with the resulting read accuracy, must be improved before it will be widely adopted and standardized technology in the field. Thus, to address the improvements needed to enable high-accuracy nanopore-based strand sequencing, EBS proposes to develop broadly applicable methodology that will vastly improve both library preparation processes and the ability to control strand translocation, which will provide significantly higher quality sequencing accuracy such that it will yield a nanopore-based technology that can be confidently used for clinical or POC use.